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LMP301 > LMP 301 Lecture 20: Molecular Diagnostics > Flashcards

LMP 301 Lecture 20: Molecular Diagnostics Flashcards

1
Q

molecular diagnosis includes…

A
  • pharmacogenetics
  • identity testing (forensics)
  • molecular genetics
  • hematology
  • molecular oncology
  • infectious diseases
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2
Q

what is pharmacogenetics?

A
  • personalized medicine

- prescribe drugs to person based on their genome

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3
Q

what type of disorder is sickle cell anemia (genetics)?

A

Autosomal recessive

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4
Q

what part of the genome has a problem in those with sickle cell anemia?

A

single point mutation in B-globulin gene

  • E -> V
  • restriction enzyme (MstII) is no longer able to recognize the sequence that it’s supposed to digest
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5
Q

MstII cuts between…

A

C and T

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6
Q

RFLP

A

restriction fragment length polymorphism

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7
Q

RFLP is used to…

A
  1. digest the genome with restriction enzymes
  2. use southern blot to detect length of digested pieces
  3. compare to normal to see if there is mutation / what the mutation affects
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8
Q

what differs in a mutant phenotype when looking at RFLP results?

A

usually, some sites are not cut (no longer recognized by the restriction enzyme). This will lead to larger segments when compared with the normal phenotype

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9
Q

How long does southern blot take?

A

about 1 week

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10
Q

process of southern blot (RFLP method)

A
  1. restriction enzyme digest sample
  2. electrophoresis on sample
  3. immobilization
  4. hybridization
  5. detection
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11
Q

limitations of southern blot based on RFLP method

A
  • labour intensive
  • time consuming / slow turn around time
  • require operator skills / not automated
  • use radioactive isotopes
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12
Q

steps of PCR

A
  1. denature DNA (95*C)
  2. anneal primers (55*C)
  3. extension - Taq adds nucleotides (72*C)
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13
Q

30 cycles of PCR can produce…

A

1 billion PCR products (new strands)

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14
Q

PCR allows us to look at target gene without…

A

highly sensitive probes (because there’s so many!)

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15
Q

turn-around time of PCR-RFLP

A

1-2 days

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16
Q

process of PCR-RFLP

A
  1. PCR reaction
  2. Restriction enzyme digestion
  3. Gel electrophoresis
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17
Q

limitations with PCR-RFLP

A
  • based on known sequence (get right primer & restriction enzymes)
  • risk of contamination (will amplify mistakes)
  • miss heterozygous large insertion/deletion
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18
Q

what technique is used to examine multiple mutations associated with a disease?

A

multiplexed PCR-RFLP

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19
Q

multiplexed PCR-RFLP

A
  • use multiple primers to amplify several DNA fragments in 1 run
  • genotype based on electrophoresis pattern
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20
Q

what disease is genotyped using multiplexed PCR-RFLP?

A

Hereditary hemocromatosis (HH)

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21
Q

which gene is affected in those with HH?

A

HFE

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22
Q

mutations of CF

A

many mutations in the disease-causing gene

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23
Q

effect of CF / why is it named this way?

A

scarring (fibrosis) and cyst formation in the pancreas

24
Q

what is the most common life-limiting autosomal recessive disease among Caucasians?

A

CF

25
Q

what type of disease is CF (genetics)

A

autosomal recessive

26
Q

incidence of disease

A

1 in 2500

27
Q

incidence of carrier for CF

A

1 in 25

28
Q

what is the affected gene in CF?

A

cystic fibrosis transmembrane conductance regulator (CFTR)

29
Q

CFTR codes…

A

protein which is responsible for transport of Cl- ions across membrane (lungs, pancreas, liver, digestive tract, reproductive tract, skin)

30
Q

CFTR is a ___ transporter

A

ABC (ATP-binding cassette)

31
Q

what happens if there is defective CFTR?

A

thick, viscous, mucus secretions

32
Q

onset of CF symptoms happen…

A

at birth or later in childhood

33
Q

symptoms of CF

A
  • frequent lung infection (due to mucus secretions)
  • poor growth (malnutrition due to mucus in GI)
  • infertility
  • diabetes
34
Q

CFTR has __ exons and span more than __ kb

A

27

230

35
Q

CFTR gene is located on chromosome __

A

7

36
Q

how many mutations are there for the CFTR gene?

A

more than 1600

37
Q

purpose of genetic screening

A

diagnose affected patients and identify carriers

38
Q

how many mutations CFTR mutations are screened for? why were they picked?

A

25; >0.1% frequency in US population

39
Q

what are the most common mutations in CFTR?

A

F508: deletion for Phe at position 508

40
Q

Platforms for CFTR mutations

A
  • Tag-It mutation detection system
  • Denaturing high performance liquid chromatography (DHPLC)
  • non-PCR based technologies (Third Wave INVADER)
41
Q

Tag-It mutation detection system

A
  • bead-based microarray platform
  • many primers that recognize specific sequences (mutations)
  • catch about 50 mutations
  • count beats to see if wild type or mutatnt
42
Q

DHPLC

A
  • adjust temp

- mismatches separate at lower temp than the right match

43
Q

DNA sequencing

A

determines precise order of nucleotides in a DNA molecule

- any method/technology that determines the order of the bases

44
Q

4 basic sequencing methods

A
  1. chemistry reaction
  2. technology
  3. labeling strategy
  4. sequencing detection
45
Q

2 chemistry reactions used to sequence

A
  1. enzymatic dideoxy (Sanger)

2. chemical (Maxam-Gilbert)

46
Q

2 technologies used to sequence

A
  1. chain termination

2. thermal cycle

47
Q

2 labeling strategies used to sequence

A
  1. primers

2. dideoxynucleotides

48
Q

2 sequence detection methods used to sequence

A
  1. radioactive

2. fluorescent

49
Q

what is the gold standard for mutation detection & confirmation (sequencing)?

A

Sanger sequencing

50
Q

Sanger sequencing

A
  • use DNA Pol to make a copy of ssDNA template at the 3’ end of a primer
  • use PCR
  • terminate by adding 2’, 3’-dideoxynucleotides
  • analyze fragment using gel/capillary electrophoresis
51
Q

benefits of Sanger DNA sequencing

A
  • automated (capillary electrophoresis)
  • CCD laser detector
  • multicolour fluorescent labeling & detection
  • many applications for data (sequencing, fragment analysis, genotyping & SNP discovery, microsatellite analysis)
  • software for data collection & analysis
52
Q

how does Sanger sequencing method terminate?

A

Add 2,3-dideoxyribose dideoxynucleodies

  • li H instead of OH at C3
  • won’t H bond with the next nucleotide, so chain stops
53
Q

limitations of Sanger sequencing

A
  • miss heterozygous large insertion/deletion
  • cost, labour, turn-around time
  • difficult to interpret variants with unknown clinical significance (variation in genome or harmful mutation?)
54
Q

clinical applications of DNA sequencing

A
  • detect mutations
  • confirm mutations detected by other methods
  • genotyping (histocompatibility typing, resistance testing)
55
Q

histocompatibility typing

A

used for transplant patients to see if organ is compatible

LMP301 (18 decks)

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